Biochemical characterization of Bombyx mori Dicer-2 that dices double-stranded RNAs into 20-nt small RNA.

We detected enzymatic activity that generates 20-nucleotide (nt) RNA from double-stranded RNAs (dsRNAs) in crude extracts prepared from various silkworm (Bombyx mori) organs. The result using knocked-down cultured cells indicated that this dicing activity originated from B. mori Dicer-2 (BmDcr2). Biochemical analyses revealed that BmDcr2 preferentially cleaves 5'-phosphorylated dsRNAs at the 20-nt site-counted from the 5'-phosphorylated end-and required ATP and magnesium ions for the dicing reaction. This is the first report of the biochemical characterization of Dicer-2 in lepidopteran insects. This enzymatic property of BmDcr2 in vitro is consistent with the in vivo small interfering RNA profile in virus-infected silkworm cells.

Caenorhabditis elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA.

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

Inducible deletion of microRNA activity in kidney mesenchymal cells exacerbates renal fibrosis.

MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-ß (PDGFR-ß)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-ß. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-ß. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.

Caspase-mediated processing of TRBP regulates apoptosis during viral infection.

RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection.

Deep mutational scanning of the RNase III-like domain in Trypanosoma brucei RNA editing protein KREPB4.

Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degron-controlled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages.

Non-canonical RNA substrates of Drosha lack many of the conserved features found in primary microRNA stem-loops.

The RNase III enzyme Drosha has a central role in microRNA (miRNA) biogenesis, where it is required to release the stem-loop intermediate from primary (pri)-miRNA transcripts. However, it can also cleave stem-loops embedded within messenger (m)RNAs. This destabilizes the mRNA causing target gene repression and appears to occur primarily in stem cells. While pri-miRNA stem-loops have been extensively studied, such non-canonical substrates of Drosha have yet to be characterized in detail. In this study, we employed high-throughput sequencing to capture all polyA-tailed RNAs that are cleaved by Drosha in mouse embryonic stem cells (ESCs) and compared the features of non-canonical versus miRNA stem-loop substrates. mRNA substrates are less efficiently processed than miRNA stem-loops. Sequence and structural analyses revealed that these mRNA substrates are also less stable and more likely to fold into alternative structures than miRNA stem-loops. Moreover, they lack the sequence and structural motifs found in miRNA stem-loops that are required for precise cleavage. Notably, we discovered a non-canonical Drosha substrate that is cleaved in an inverse manner, which is a process that is normally inhibited by features in miRNA stem-loops. Our study thus provides valuable insights into the recognition of non-canonical targets by Drosha.

let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching.

ABSTRACT: The switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (ß-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A-mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA-mediated inhibitory pathway (let-7 ⊣ HIC2 ⊣ BCL11A ⊣ HBG).

Environmental arsenic pollution induced liver oxidative stress injury by regulating miR-155 through inhibition of AUF1.

Arsenic (As), a common environmental pollutant, has become a hot topic in recent years due to its potentially harmful effects. Liver damage being a central clinical feature of chronic arsenic poisoning. However, the underlying mechanisms remain unclear. We demonstrated that arsenic can lead to oxidative stress in the liver and result in structural and functional liver damage, significantly correlated with the expression of AUF1, Dicer1, and miR-155 in the liver. Interestingly, knockdown AUF1 promoted the up-regulatory effects of arsenic on Dicer1 and miR-155 and the inhibitory effects on SOD1, which exacerbated oxidative damage in rat liver. However, overexpression of AUF1 reversed the up-regulatory effects of arsenic on Dicer1 and miR-155, restored arsenic-induced SOD1 depletion, and attenuated liver oxidative stress injury. Further, we verified the mechanism and targets of miR-155 in regulating SOD1 by knockdown/overexpression of miR-155 and nonsense mutant SOD1 3'UTR experiments. In conclusion, these results powerfully demonstrate that arsenic inhibits AUF1 protein expression, which in turn reduces the inhibitory effect on Dicer1 expression, which promotes miR-155 to act on the SOD1 3'UTR region after high expression, thus inhibiting SOD1 protein expression and enzyme activity, and inducing liver injury. This finding provides a new perspective for the mechanism research and targeted prevention of arsenic poisoning, as well as scientific evidence for formulating strategies to prevent and control environmental arsenic pollution.

Cytoplasmic and nuclear DROSHA in human villous trophoblasts.

In villous trophoblasts, DROSHA is a key ribonuclease III enzyme that processes pri-microRNAs (pri-miRNAs) into pre-miRNAs at the placenta-specific, chromosome 19 miRNA cluster (C19MC) locus. However, little is known of its other functions. We performed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq) analysis of terminal chorionic villi to identify DROSHA-binding RNAs in villous trophoblasts. In villous trophoblasts, DROSHA predominantly generated placenta-specific C19MC pre-miRNAs, including antiviral C19MC pre-miRNAs. The fCLIP-seq analysis also identified non-miRNA transcripts with hairpin structures potentially capable of binding to DROSHA (e.g., SNORD100 and VTRNA1-1). Moreover, in vivo immunohistochemical analysis revealed DROSHA in the cytoplasm of villous trophoblasts. DROSHA was abundant in the cytoplasm of villous trophoblasts, particularly in the apical region of syncytiotrophoblast, in the full-term placenta. Furthermore, in BeWo trophoblasts infected with Sindbis virus (SINV), DROSHA translocated to the cytoplasm and recognized the genomic RNA of SINV. Therefore, in trophoblasts, DROSHA not only regulates RNA metabolism, including the biogenesis of placenta-specific miRNAs, but also recognizes viral RNAs. After SINV infection, BeWo DROSHA-binding VTRNA1-1 was significantly upregulated, and cellular VTRNA1-1 was significantly downregulated, suggesting that DROSHA soaks up VTRNA1-1 in response to viral infection. These results suggest that the DROSHA-mediated recognition of RNAs defends against viral infection in villous trophoblasts. Our data provide insight into the antiviral functions of DROSHA in villous trophoblasts of the human placenta.

Two-motif model illuminates DICER cleavage preferences.

In humans, DICER is a key regulator of gene expression through its production of miRNAs and siRNAs by processing miRNA precursors (pre-miRNAs), short-hairpin RNAs (shRNAs), and long double-stranded RNAs (dsRNAs). To advance our understanding of this process, we employed high-throughput dicing assays using various shRNA variants and both wild-type and mutant DICER. Our analysis revealed that DICER predominantly cleaves shRNAs at two positions, specifically at 21 (DC21) and 22 (DC22) nucleotides from their 5'-end. Our investigation identified two different motifs, mWCU and YCR, that determine whether DICER cleaves at DC21 or DC22, depending on their locations in shRNAs/pre-miRNAs. These motifs can work together or independently to determine the cleavage sites of DICER. Furthermore, our findings indicate that dsRNA-binding domain (dsRBD) of DICER enhances its cleavage, and mWCU strengthens the interaction between dsRBD and RNA, leading to an even greater enhancement of the cleavage. Conversely, YCR functions independently of dsRBD. Our study proposes a two-motif model that sheds light on the intricate regulatory mechanisms involved in gene expression by elucidating how DICER recognizes its substrates, providing valuable insights into this critical biological process.

The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways.

In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1 expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.

Comparative Transcriptomic Analysis of Flagellar-Associated Genes in Salmonella Typhimurium and Its rnc Mutant.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a globally recognized foodborne pathogen that affects both animals and humans. Endoribonucleases mediate RNA processing and degradation in the adaptation of bacteria to environmental changes and have been linked to the pathogenicity of S. Typhimurium. Not much is known about the specific regulatory mechanisms of these enzymes in S. Typhimurium, particularly in the context of environmental adaptation. Thus, this study carried out a comparative transcriptomic analysis of wild-type S. Typhimurium SL1344 and its mutant (∆rnc), which lacks the rnc gene encoding RNase III, thereby elucidating the detailed regulatory characteristics that can be attributed to the rnc gene. Global gene expression analysis revealed that the ∆rnc strain exhibited 410 upregulated and 301 downregulated genes (fold-change > 1.5 and p < 0.05), as compared to the wild-type strain. Subsequent bioinformatics analysis indicated that these differentially expressed genes are involved in various physiological functions, in both the wild-type and ∆rnc strains. This study provides evidence for the critical role of RNase III as a general positive regulator of flagellar-associated genes and its involvement in the pathogenicity of S. Typhimurium.

Bacterial RNase III: Targets and physiology.

Bacteria can rapidly adapt to changes in their environment thanks to the innate flexibility of their genetic expression. The high turnover rate of RNAs, in particular messenger and regulatory RNAs, provides an important contribution to this dynamic adjustment. Recycling of RNAs is ensured by ribonucleases, among which RNase III is the focus of this review. RNase III enzymes are highly conserved from prokaryotes to eukaryotes and have the specific ability to cleave double-stranded RNAs. The role of RNase III in bacterial physiology has remained poorly explored for a long time. However, transcriptomic approaches recently uncovered a large impact of RNase III in gene expression in a wide range of bacteria, generating renewed interest in the physiological role of RNase III. In this review, we first describe the RNase III targets identified from global approaches in 8 bacterial species within 4 Phyla. We then present the conserved and unique functions of bacterial RNase III focusing on growth, resistance to stress, biofilm formation, motility and virulence. Altogether, this review highlights the underestimated impact of RNase III in bacterial adaptation.

USP7 reduces the level of nuclear DICER, impairing DNA damage response and promoting cancer progression.

Endoribonuclease DICER is an RNase III enzyme that mainly processes microRNAs in the cytoplasm but also participates in nuclear functions such as chromatin remodelling, epigenetic modification and DNA damage repair. The expression of nuclear DICER is low in most human cancers, suggesting a tight regulation mechanism that is not well understood. Here, we found that ubiquitin carboxyl-terminal hydrolase 7 (USP7), a deubiquitinase, bounded to DICER and reduced its nuclear protein level by promoting its ubiquitination and degradation through MDM2, a newly identified E3 ubiquitin-protein ligase for DICER. This USP7-MDM2-DICER axis impaired histone γ-H2AX signalling and the recruitment of DNA damage response (DDR) factors, possibly by influencing the processing of small DDR noncoding RNAs. We also showed that this negative regulation of DICER by USP7 via MDM2 was relevant to human tumours using cellular and clinical data. Our findings revealed a new way to understand the role of DICER in malignant tumour development and may offer new insights into the diagnosis, treatment and prognosis of cancers.

JANUS, a spliceosome-associated protein, promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) are important regulators of genes expression. Their levels are precisely controlled through modulating the activity of the microprocesser complex (MC). Here, we report that JANUS, a homology of the conserved U2 snRNP assembly factor in yeast and human, is required for miRNA accumulation. JANUS associates with MC components Dicer-like 1 (DCL1) and SERRATE (SE) and directly binds the stem-loop of pri-miRNAs. In a hypomorphic janus mutant, the activity of DCL1, the numbers of MC, and the interaction of primary miRNA transcript (pri-miRNAs) with MC are reduced. These data suggest that JANUS promotes the assembly and activity of MC through its interaction with MC and/or pri-miRNAs. In addition, JANUS modulates the transcription of some pri-miRNAs as it binds the promoter of pri-miRNAs and facilitates Pol II occupancy of at their promoters. Moreover, global splicing defects are detected in janus. Taken together, our study reveals a novel role of a conserved splicing factor in miRNA biogenesis.

Recent progress in miRNA biogenesis and decay.

MicroRNAs are a class of small regulatory RNAs that mediate regulation of protein synthesis by recognizing sequence elements in mRNAs. MicroRNAs are processed through a series of steps starting from transcription and primary processing in the nucleus to precursor processing and mature function in the cytoplasm. It is also in the cytoplasm where levels of mature microRNAs can be modulated through decay mechanisms. Here, we review the recent progress in the lifetime of a microRNA at all steps required for maintaining their homoeostasis. The increasing knowledge about microRNA regulation upholds great promise as therapeutic targets.

Quantitative Investigation of FAD2 Cosuppression Reveals RDR6-Dependent and RDR6-Independent Gene Silencing Pathways.

The frequency and extent of transgene-mediated cosuppression varies substantially among plant genes. However, the underlying mechanisms leading to strong cosuppression have received little attention. In previous studies, we showed that the expression of FAD2 in the seeds of Arabidopsis results in strong RDR6-mediated cosuppression, where both endogenous and transgenic FAD2 were silenced. Here, the FAD2 strong cosuppression system was quantitatively investigated to identify the genetic factors by the expression of FAD2 in their mutants. The involvement of DCL2, DCL4, AGO1, and EIN5 was first confirmed in FAD2 cosuppression. SKI2, a remover of 3' end aberrant RNAs, was newly identified as being involved in the cosuppression, while DCL3 was identified as antagonistic to DCL2 and DCL3. FAD2 cosuppression was markedly reduced in dcl2, dcl4, and ago1. The existence of an RDR6-independent cosuppression was revealed for the first time, which was demonstrated by weak gene silencing in rdr6 ein5 ski2. Further investigation of FAD2 cosuppression may unveil unknown genetic factor(s).

Pri-miRNA cleavage assays for the Microprocessor complex.

The Microprocessor complex (MP) is a vital component in the biogenesis of microRNAs (miRNAs) in animals. It plays a crucial role in the biogenesis of microRNAs (miRNAs) in mammals as it cleaves primary miRNAs (pri-miRNAs) to initiate their production. The accurate enzymatic activity of MP is critical to ensuring proper sequencing and expression of miRNAs and their correct cellular functions. RNA elements in pri-miRNAs, including secondary structures and sequencing motifs, RNA editing and modifications, and cofactors, can impact MP cleavage and affect miRNA expression and sequence. To evaluate MP cleavage activity with various RNA substrates under different conditions, we set up an in vitro pri-miRNA cleavage assay. This involves purifying human MP from HEK293E cells, synthesizing pri-miRNAs using in vitro transcription, and performing pri-miRNA cleavage assays using basic laboratory equipment and reagents. These procedures can be performed in various labs and improved for high-throughput analysis of enzymatic activities with thousands of RNA substrates.

The pre-miRNA cleavage assays for DICER.

MicroRNAs (miRNAs) are small, non-coding RNA molecules that play a crucial role in gene silencing. The gene-silencing activity of miRNAs depends on their sequences and expression levels. The human RNase III enzyme DICER cleaves miRNA precursors (pre-miRNAs) to produce miRNAs, making it crucial for miRNA production and cellular miRNA functions. DICER is also critical for the gene silencing technology using short-hairpin RNAs (shRNAs), which are cleaved by DICER to generate siRNAs that knockdown target genes. The DICER cleavage assay is an important tool for investigating its molecular mechanisms, which are essential for understanding its functions in miRNA biogenesis and shRNA-based gene silencing technology. The assay involves DICER protein purification, preparation of pre-miRNA and shRNA substrates, and the cleavage assay, using common molecular biology equipment and commercialized reagents that can be applied to other RNA endonucleases.

Profiling the in vivo RNA interactome associated with the endoribonuclease RNase III in Staphylococcus aureus.

Regulatory small RNA (sRNA) have been extensively studied in model Gram-negative bacteria, but the functional characterisation of these post-transcriptional gene regulators in Gram-positives remains a major challenge. Our previous work in enterohaemorrhagic E. coli utilised the proximity-dependant ligation technique termed CLASH (UV-crosslinking, ligation, and sequencing of hybrids) for direct high-throughput sequencing of the regulatory sRNA-RNA interactions within the cell. Recently, we adapted the CLASH technique and demonstrated that UV-crosslinking and RNA proximity-dependant ligation can be applied to Staphylococcus aureus, which uncovered the first RNA-RNA interaction network in a Gram-positive bacterium. In this chapter, we describe modifications to the CLASH technique that were developed to capture the RNA interactome associated with the double-stranded endoribonuclease RNase III in two clinical isolates of S. aureus. To briefly summarise our CLASH methodology, regulatory RNA-RNA interactions were first UV-crosslinked in vivo to the RNase III protein and protein-RNA complexes were affinity-purified using the His6-TEV-FLAG tags. Linkers were ligated to RNase III-bound RNA during library preparation and duplexed RNA-RNA species were ligated together to form a single contiguous RNA 'hybrid'. The RNase III-RNA binding sites and RNA-RNA interactions occurring on RNase III (RNA hybrids) were then identified by paired-end sequencing technology. RNase III-CLASH represents a step towards a systems-level understanding of regulatory RNA in Gram-positive bacteria.